Cryoprotective aqueous solutions useful for the preservation of in vitro cultured epithelial sheets

ABSTRACT

A method for the preservation of in vitro expanded epithelial sheets by incubation of the sheets in a cryopreserving solution characterized by lack of volume increase at the liquid-solid phase transition. The cryopreserving solutions comprise: a) polyethyleneglycols having molecular weight between 3 and 5 KD; and b) at least one cross-linking agent selected from polyols, polyamines, mono- or oligosaccharides, and polyethyleneglycols having molecular weight lower than 1 KD.

BACKGROUND OF THE INVENTION

This application is a 371 of PCT/EP93/03364 filed Dec. 1, 1993.

The present invention refers to the preservation, by freezing, ofepithelium sheets obtained from cell cultures of keratinocytes.

The cultures of human keratinocytes, developed by. Green and Rheinwald(U.S. Pat. Nos. 4,016,036; 4,304,865; 4,456,687) raise more and moreinterest in several medical applications such as in the treatment ofserious burns, ulcers, difficult to heal scars and in plasticoncological and reconstructive surgery.

In the case of seriously burnt patients, the death-rate, depending onthe patient's age and affected body surface, is dramatically high,particularly for third degree burns greater than 60%. According to theusual procedure, the damaged body surfaces are covered by transplantingskin films removed from healthy areas (auto-grafts); this procedure isvery time-consuming and cannot be practically used when the burnt areaexceeds 60% of the body surface. The technique by Rheinwald and Greenhas allowed the burnt areas to be covered with autologous epithelialfilms obtained from the patient's epithelial cells (keratinocytes).Starting from a small biopsy of epithelial tissue, the technique affordsan epithelium surface sufficient to cover the whole body surface in afew weeks.

The autologous transplant in seriously burnt patients represents aunique, socially relevant therapeutic approach.

Autologous transplants are carried out also in cases of plasticoncological and reconstructive surgery. In this case, before surgery,sheets of epithelium expanded in vitro are prepared starting from apatient's biopsy.

The use of cultured human epithelium sheets as "living bandage", for thetreatment of ulcers and scars which are difficult to heal, isparticularly interesting not only from the social but also from thecommercial point of view. In this case, the epidermis sheet, optionallyobtained from suitably typized keratinocytes cultures, is used as atemporary dressing of the scar, making the restoring of the epitheliumthereon easier thanks to the production of a series of factorsstimulating the growth of this tissue, decreasing therefore thepatient's suffering and hospitalization.

More than 4 million patients could be treated in the United States withthis new therapeutic approach. It is obvious however that this wouldrequire remarkably reduced costs of the epithelium sheets, long and easypreservability of the product and easy application procedures inhospital environments.

The method originally developed by Green and Rheinwald for autograftscomprises the sampling of a small fragment (2-3 cm²) of epithelium froma healthy area, from which an epithelial cellular suspension is obtainedand cultured and finally expanded by means of subsequent passages of theprimary cultures. When the secondary and tertiary cultures becomeconfluent, multilayered epithelium sheets are obtained in differentsizes according to the need.

Notwithstanding the high potentiality of this therapeutic approach, itswide-scale application is limited by the requirement that the obtainedepithelial sheets should be used within a few hours after theirpreparation.

EP-A-0296475 discloses a method for preserving at -100° C. viable,transplantable epithelium sheets cultured in vitro, frozen in a nutrientmedium containing the usual cryopreserving agents such asdimethylsulfoxide or glycerine.

Although this method has allowed for the first time the preservation ofin vitro cultured epithelium sheets, a number of problems limit itsindustrial applicability:

a) the permanence in the preserving medium before freezing is extremelycritical for the cells at room temperature. This makes critical theinitial step of the preserving process, comprising an incubation at roomtemperature to equilibrate the epithelium sheets with the solution andthe final phase of thawing before using the sheets. In both cases, a fewminutes difference in the performance of these steps may (irreparably)impair the characteristics of the product;

b) The complexity of the cooling cycle of the samples, criticallycharacterized by different cooling rates in particular temperatureranges involve expensive industrial apparatuses and high productioncosts;

c) The preserved product is very sensitive to even slight changes in thepreservation temperature, which can cause alteration of the productpreventing its use;

d) The shipment of the sample to the utilization sites must necessarilybe carried out at a temperature constantly kept at about -80° C. andsimilar measures must be observed by the hospitals before the use of theproduct;

e) Cell viability in the frozen graft obtained by this procedure can be,in the best conditions, up to 70%. However given the critical stepsrequired for freezing (see points a, b, c, d) the large variability ofthe obtained results makes this procedure difficult to use in largescale preparations.

The present invention aims at overcoming the above limitations andallowing the industrial applicability of the production and preservationprocesses of human epithelium sheets optionally typized, convenientmethods of use in hospital environments and costs compatible with thepreviously cited therapeutic uses.

BRIEF DESCRIPTION OF THE INVENTION

The above problem is solved by the present invention which provides anew kind of cryoprotective solutions which allow the preserving offrozen cultured human epithelial sheets for a long time.

According to the invention, the processing times are not critical bothbefore freezing and in hospitals where thawing and grafting take place.

Moreover, the quality of the graft frozen with the new procedure is muchsuperior in cell viability which can be up to 100% in the bestconditions. At variance with grafts frozen with the old procedure thefrozen sheets, preserved in these new cryoprotective solutions, do notundergo functional alterations impairing their use, even if thepreservation temperature is subjected to changes of tens of degrees orthe sample is accidentally thawed for a short period of time.

It is known that in the cryopreservation of biological material inaqueous solutions, the formation of ice crystals, connected with theregular growth of the crystalline reticulum, brings about a high risk ofmechanical destruction of cell structures situated along the path ofcrystal growth. This occurs both outside the cells, in the solutionswhere they are placed for the freezing phase, and inside the cellsthemselves. This drawback is only partially overcome by usingcryopreserving agents such as glycerine, dimethylsulfoxide etc.

The very high pressures acting on the cell structures when water passesfrom the liquid to the solid state act synergistically with themechanical damage deriving from the formation of ice needles. The volumeincrease for aqueous solutions is up to 10%.

Both the crystal formation and the volume change at the liquid-solidtransition phase usually occur in the solutions presently used for thecryopreservation of biological material and are the main cause of theviability loss of the cryopreserved samples. The present inventionprovides cryopreserving solutions in which the liquid-solid transitionphase occurs without volume increase and macrocrystal formation,providing therefore improvements over the prior-art methods and solvingin an economic and convenient way the many problems of thecryopreservation of biological material, particularly those previouslycited for the preservation of frozen epithelial tissue sheets.

The solutions of the invention comprise:

a) polyethylene glycols (PEG) having molecular weight not higher than 20KD, preferably from 3 to 5 KD, at concentrations not higher than 25%w/w, preferably from 10 to 20% w/w;

b) at least one low molecular weight compound, defined as across-linking agent in view of its ability to form with the polymer andwith water cross-linked systems based on hydrogen bends, inconcentrations not higher then 25% w/w, preferably from 5 to 20% w/w.The cross-linking agents are selected from polyols (glycerine, ethyleneglycols, maltitel, inositol), mono and oligosaccharides (glucose,sucrose, maltodextrins) and PEGs having molecular weight lower than 1KD. Glycerine is preferably used.

The solutions of the invention may be prepared in physiological salinesolutions and may contain antibiotics, proteins, sera and othercompounds usually used for the growth and culture of biologicalmaterials.

More particularly, solutions suited for the cryopreservation of viable,in vitro grown epithelium sheets are prepared by adding in suitableconcentrations PEG and glycerol as cross-linking agent, preferably 13%w/v PEG an 15% w/v glycerol to media optimized for the keratinocytesgrowth such as 60% v/v Dulbecco modification of Eagle's medium, 30% v/vHam's F12, 10% v/v Fetal calf serum (FCS), containing glutamine 4 mM,adenine 1.8×10⁻⁴ M, insulin 5 μg/mL, transferrine 5 μg/mL,triiodothyronina 2×10⁻⁹ M hydrocortisone 0.4 μg/mL,penicillin-streptomycine 50 U/mL.

The sheets are first washed in the cryopreserving solution and thenintroduced into suitable bags containing 100-200 ml of solution.

After closing the containers, the samples are allowed to equilibrate atroom temperature for periods not longer than 1 h, preferably from 5 to10 min.

Freezing of samples may be carried out by simply cooling the containersto the preservation temperature, generally from -25 to -100° C.,preferably -80° C.

Thawing of samples may be carried out by dipping the frozen container ina bath at 37° C. for 5-10 min. In order to make this step less critical,especially when not particularly qualified users have to handle suchdelicate cellular materials, and to minimize stress to which thawedcells may be subjected by the high concentration of cryoprotectiveagents in the liquid phase, thawing of epithelial sheets may be carriedout in a special container consisting of two compartments which areinterconnected when the sample is thawed, one containing the epithelialsheets in the cryopreserving solution and the other, containing anutrient medium or isotonic saline solution. During thawing, the twosolutions are mixed, causing the dilution of cryoprotective agentsminimizing thereby possible cellular damage.

The human epithelial sheets cryopreserved in the solutions of theinvention are equivalent to the fresh ones not subjected to freezing,both as far as the morphology and the after graft are concerned.

The solutions of the invention may also be used for the preservation ofcells used for the preparation of the sheets.

These comprise, for instance, 3T3 cells to be used as feeder layer orkeratinocytes isolated and dissociated from biopsies.

The following examples further illustrate the invention.

EXAMPLE 1

Keratinocytes cultures were obtained according to the method ofRheinwald and Green. The secondary confluent keratinocytes culturesobtained according to U.S. Pat. No. 4,016,036 are detached from cultureflasks, transferred on suitable support and anchored by means of metalclips for vascular surgery according to the method of U.S. Pat. No.4,304,866.

The so prepared sheets are washed by immersion in sterile conditions in100 ml of the cryopreserving solution consisting of 60% v/v Dulbeccomodification of Eagle's medium, 30% v/v Ham's F12, 10% v/v FCS,glutamine 4 mM, adenine 1.8×10⁻⁴ M, insuline 5 μg/mL, transferrine 5μg/mL, triodothyrosine 2×10⁻⁹ M, hydrocortisone 0.4 μg/mL, epidermalgrowth factor (EGF) 10 ng/mL, penicilline-streptomicine 50 U/mL, PEG3350 13% p/v, glycerol 15% p/v.

The sheets are then transferred under sterile conditions in 15×10 cmplastic envelopes, previously sterilized and containing 100 ml of theabove reported cryopreserving solution.

The plastic envelopes are then heat sealed and then transferred, withoutany particular precaution, to the freezer and cooled to a temperatureranging from -25° and -100° C., preferably -80° C. The duration of theoperations bringing the epithelial sheets into contact with thecryopreserving solution at room temperature is not critical and mayrange from a few minutes to 1 h, preferably 5-10 min, without negativeconsequences in the clinical use of the preserved material.

The liquid-solid transition phase of the solution occurs between -10°and -15° C. and the cooling rate of the samples is not critical and thepreserving temperature may vary even by tens of degrees withoutaffecting viability of the preserved material.

The epithelial sheets may be preserved at -80° C. for several months,whereas at higher temperatures this period is slightly shortened. Theenvelopes containing the frozen epithelial sheets may be shipped insolid CO₂ or at higher temperatures up to -20° C. and the therapeuticuse is not impaired by temperature changes of tens of degrees or by ashort accidental thawing.

Immediately before use, the envelopes, taken out of the freezer areimmediately introduced into a water bath at 37° C. for 5-10 min and thenimmersed for a few seconds in 70% ethanol. After opening in sterileconditions, the epithelial sheets are transferred into sterilecontainers, thoroughly washed with a medium free from serum andadditives or with saline solution before use.

The thawed samples are subjected to the following characterizations:

a) histology of the sheet, morphological exam and evaluation of thekeratins typical of the various differentiative degrees;

b) trypsinization and low density plating to evaluate the number andsize of the formed colonies;

c) microscopic evaluation of the cellular morphology and of the numberof viable cells;

d) biological tests of graftability on derma or other vital receivingbed, such as nude mice.

The overall results of these characterizations show that cryopreservedepithelial sheets in the solutions of the invention remain viable andshow characteristics comparable to those of fresh sheets which have beennot cryopreserved.

EXAMPLE 2

Epithelial sheets cryopreserved at -80° C. for 30 days, as disclosed inExample 1, are thawed by immersing for 5-7 min the container in a bathat 37° C. and, after opening in sterile conditions, the epithelialsheets are washed in 100 ml of culture medium consisting of 60% v/vDulbecco modification of Eagle's medium, 30% v/v Ham's F12, 10% v/v FCS,glutamine 4 mM, adenine 1.8×10⁻⁴ M, insuline 5 μg/mL, transferrine 5μg/mL, triodothyronine 2×10⁻⁹ M, hydrocortisone 0.4 μg/mL, EGF 10 ng/mL,penicillin-streptomycine 50 U/mL.

The sheets are then treated with trypsine and dissociated into singlecells which are cultivated on a bed of irradiated fibroblasts, asdisclosed in U.S. Pat. No. 4,016,036. When keratinocytes colonies aresubconfluent the culture medium is removed, the cell layer is washedwith FCS-free culture medium and then treated with trypsine todissociate colonies into single cells.

The obtained cellular suspension is centrifuged, suspended again in 5 mlof cryoprotective solution, centrifuged again at 1000 g for 5 min, thesolution is removed, the cell pellet is suspended in 1.5 ml ofcryoprotective solution and frozen to -80° C. in three aliquots. Atdifferent time intervals, ranging from week to 3 months the cells arethawed by placing the closed vial for 5-7 min in a bath at 37° C., 5 mlof fresh medium are added, the suspension is centrifuged at 1000 g for 5min and suspended again in the medium with which the culture onirradiated fibroblasts is started, as reported in U.S. Pat. No.4,016,036 until a multi-layer epithelium is obtained on which thefollowing characterizations are carried out:

a) histology of the sheet, morphological exam and evaluation of thekeratins typical of the various differentiative degrees;

b) trypsinization and low density plating to evaluate the number andsize of the formed colonies;

c) microscopic evaluation of the cellular morphology and of the numberof viable cells;

d) biological tests of graftability on derma or other vital receivingbed, such as nude mice.

The results of these characterizations show that the cells deriving fromepithelium sheets cryopreserved in the solutions of the invention, afterenzymatic treatment and freezing, remain perfectly viable and keep theircharacteristics and differentiative features, as shown by the ability ofyielding again epithelial sheets fully comparable to sheets obtainedfrom non frozen cells.

We claim:
 1. Cryoprotective aqueous solutions for preservation of invitro cultured epithelial sheets comprising:a) from 10 to 20% w/w ofpolyethyleneglycols having molecular weight from 3 to 5 KD; b) from 5 to25% w/v of at least one cross-linking agent selected from the groupconsisting of polyols, polyamines, mono- and oligosaccharides, andpolyethyleneglycols having a molecular weight lower than 1 KD;andwherein a) and b) are in proportions sufficient to provide acryoprotective aqueous solution for epithelial sheets.
 2. Solutionsaccording to claim 1 wherein the cross-linking agent b) is selected fromthe group consisting of glycerol, maltitol, glucose, fructose, sucroseand maltodextrins.
 3. Solutions according to claim 1 also containing oneor more of antibiotics, proteins and sera.
 4. Solutions according toclaim 1 which include an added media comprising 60% v/v Dulbeccomodification of Eagle's medium, 30% v/v Ham's F12, and 10% v/v Fetalcalf serum (FCS) comprised of glutamine 4 mM, adenine 1.8×10⁻⁴ M,insuline 5 μg/mL, transferrine 5 μg/mL, triiodothyronina 2×10⁻⁹ M,hydrocortisone 0,4 μg/mL, Epidermal Growth Factor (EGF) 10 ng/mL andpenicillin-streptomycine 50 U/mL.
 5. A method for the cryopreservationof epithelial sheets obtained from keratinocytes cultures comprising:a)incubation at room temperature of said epithelial sheets in acryopreserving solution of claim 1, for a time shorter than 1 h, and b)freezing the epithelial sheets in said solution at temperatures from-100° to -25° C.
 6. A method for the preservation of cells used for thepreparation of epithelial sheets expanded in vitro comprising:a)incubation at room temperature of said cells in a cryopreservingsolution of claim 1, for a time shorter than 1 h, and b) freezing theepithelial sheets in said solution at temperatures from -100° to -25° C.